CN106854635A - A kind of method that false-yellowflower milkwort root or herb cell culture method prepares saponins compound - Google Patents
A kind of method that false-yellowflower milkwort root or herb cell culture method prepares saponins compound Download PDFInfo
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Abstract
The present invention relates to a kind of method that false-yellowflower milkwort root or herb cell culture method prepares saponins compound, comprise the following steps：（1）The acquisition of false-yellowflower milkwort root or herb seed；（2）The sterilization of false-yellowflower milkwort root or herb seed and the acquisition of aseptic seedling；（3）Induction false-yellowflower milkwort root or herb Callus formation；（4）False-yellowflower milkwort root or herb callus is carried out into squamous subculture, the callus of loose stabilization is obtained；（5）Callus is transferred in fluid nutrient medium carries out shaken cultivation；（6）The inducible factor of addition variety classes and various concentrations, filters out high yield cell；（7）High yield suspension cell is carried out into Multiplying culture；（8）The drying and crushing of callus or suspension cell；（9）The extraction of total saposins, purifying and measure in callus or suspension cell.The present invention can produce the secondary metabolite of false-yellowflower milkwort root or herb, and low production cost is not limited, cycle is short by season and environment.
Description
Technical field
The present invention relates to a kind of method that suspension cell culture method prepares bio-pharmaceutical, specifically one kind utilizes chrysanthemum
The method that omei meadowrue herb callus or suspension cell produce saponins compound, belongs to plant biotechnology field.
Background technology
False-yellowflower milkwort root or herb（Polygala fallaxDunn）Category Polygalaceae rogation flower, shrub or dungarunga, long greatly
Yellow raceme is extremely beautiful, is distributed in Hunan, Fujian, Guangxi, Jiangxi, Guangdong and Yunnan, is born under the sparse woods of hillside or ditch
In paddy jungle.False-yellowflower milkwort root or herb belongs to precious Chinese herbal medicine, there is laudatory titles such as " Companumoea root " " chrysanthemum ginsengs ", and its root can be nourished, strong, wind-dispelling
Wet and easypro channels and collaterals, control physically weak after being ill, lumbar muscle strain, rheumatic arthritis, traumatic injury, acute, chronic hepatitis, metroptosis and menstruation
It is uncomfortable.False-yellowflower milkwort root or herb in the early stage understanding for just having a medical value among the people, in recent years because wild resource is ceaselessly exploited and makes
With the increasingly emphasis to its medical value, market demand gradually increases, and cause becomes to be in great demand in medicinal material market in recent years and plants
Class, price constantly goes up.And cultivate at present mainly with seminal propagation, seed production is low, and cutting propagation survival rate is also low, receives
Difficulty is obtained, wild resource is extremely limited, cause resource wretched insufficiency.Thus, build a kind of relatively reasonable, perfect chrysanthemum pouring
Lotus tissue culture system, extensive quick production false-yellowflower milkwort root or herb nursery is extremely urgent.With plant organ, tissue, colony-formation assays
Evoked callus further induce neomorph or embryoid and the quick breeding that forms plant can improve plant products, change
Kind plant quality, makes the rapid popularizing planting of improved seeds, is that sustainable exploitation utilization plant lays scientific basic.
Saponin(e is distributed mainly in the higher plant of land, is also present on a small quantity in the marine organisms such as starfish and sea cucumber.It is many
The principle active component of Chinese herbal medicine such as ginseng, polygala, balloonflower root, Radix Glycyrrhizae, the wind-weed and radix bupleuri etc. all contains saponin(e.Some saponin(es also have
There is the valuable bioactivity such as the active or antipyretic, calm of antibacterial, anticancer.False-yellowflower milkwort root or herb plant is rich in saponins material,
Influence can be produced on immunologic function, with effects such as resisting stress, anti-inflammatory, promoting blood circulation, tune fat, suppression external to hepatitis B.
It is plant biological skill that this life metabolite useful to the mankind is obtained using plant cell, tissue or organ culture
The study hotspot in art field, its great advantage is can be carried out under manual control condition completely, not by geographical environment and season
Deng the limitation of factors, the generation of super whole plant yield can be obtained by changing condition of culture and the excellent cultivating system of selection
Thank to product.False-yellowflower milkwort root or herb secondary metabolite is produced using the mass propgation of plant cell or callus, can be industrialized
Production false-yellowflower milkwort root or herb saponins compound, therefore it provides one kind is by cultivating false-yellowflower milkwort root or herb callus or suspending thin
The method of born of the same parents has great significance for the exploitation that polygalic acid prepares new way.
The content of the invention
For the above-mentioned technical problem of prior art, it is an object of the invention to provide a kind of false-yellowflower milkwort root or herb cell culture method
The method for preparing saponins compound, obtained saponins compound is can be with the saponins compound of hyoscine, so to solve
Technical problem certainly is the culture technique of the callus or suspension cell for utilizing false-yellowflower milkwort root or herb, is healed by false-yellowflower milkwort root or herb
The induction of injured tissue, squamous subculture, suspension cell culture production saponins compound, the method are not limited by season and breeding time
System, convenient, easy to operate, stability is strong.
To reach above-mentioned purpose, the present invention is achieved by the following technical solutions：
A kind of method that false-yellowflower milkwort root or herb cell culture method prepares saponins compound, comprises the following steps：
（1）The acquisition of false-yellowflower milkwort root or herb seed；
（2）The sterilization of false-yellowflower milkwort root or herb seed and the acquisition of aseptic seedling；
（3）Induction false-yellowflower milkwort root or herb Callus formation；
（4）False-yellowflower milkwort root or herb callus is carried out into squamous subculture, the callus of loose stabilization is obtained；
（5）Callus is transferred in fluid nutrient medium carries out shaken cultivation；
（6）The inducible factor of addition variety classes and various concentrations, filters out high yield cell；
（7）High yield suspension cell is carried out into Multiplying culture；
（8）The drying and crushing of callus or suspension cell；
（9）The extraction of total saposins, purifying and measure in callus or suspension cell.
The step（2）Middle false-yellowflower milkwort root or herb aseptic seedling is comprised the following steps：Fruit is cut with scalpel, is selected
Uncracked fruit is taken, with alcohol-pickled fruit 40-50 seconds, then aseptic water elution is used, quality percent by volume is subsequently transferred to
Concentration, with aseptic water washing, uses aseptic filter paper suck dry moisture to be soaked 10-12 minutes in 0.1% mercuric chloride solution, in aseptic behaviour
Make to be removed the pericarp of fruit with aseptic operation knife on platform, seed is eliminated to be placed on aseptic filter paper, then by outside seed
Plant skin to remove, the seed of immature seed and maturation is separated, then immature seed accesses MS+0.2-0.5mgL-1
Benayl aminopurine+sucrose 30gL-1+ agar 5.5gL-1Culture medium in, ripe seed is accessed in MS culture mediums, and pH is
5.8-6.0, is placed in 12hd-1Photoperiod, 50 μm of olm of illumination-2 s-1, 25 ± 1 DEG C of temperature culturing room in cultivate, mature seed 10
It or so starts to sprout, and plumular axis and radicle are grown, and rear seedling is grown up within 30 days or so, and immature seed forms adventitious bud in 7 days or so,
Adventitious bud clump is grown within 20 days or so, breeding coefficient is about 8-10.
The culture of false-yellowflower milkwort root or herb callus and subculture are comprised the following steps：With the tender leaf of obtained aseptic seedling, plumular axis, stem
The organs such as point are explant, and it is explant on inducing culture that described callus is each position of false-yellowflower milkwort root or herb aseptic seedling
Culture can obtain callus for 30 days, and the culture medium of callus induction is MS+1.0 mgL-12,4 dichlorophenoxyacetic acid+
0.1mg·L-1N- phenyl-N ' -1,2,3- thiadiazoles -5- ureas+sucrose 30gL-1+ agar 5.5gL-1, pH is 5.8-6.0,
After cultivating stabilization in the callus same medium that will be obtained, average 3 weeks subcultures once, and squamous subculture 4-6 generations, can
Obtain pale yellow, loose callus.
False-yellowflower milkwort root or herb cell suspension cell culture is comprised the following steps：The callus of acquisition is inoculated in Liquid Culture
In the triangular flask of base, culture medium is：MS+0.5 mg·L-1 2,4 dichlorophenoxyacetic acid+0.1mgL-1Benayl aminopurine+sucrose
30g·L-1+ agar 5.5gL-1, pH is 5.8-6.0, and inducible factor is added in fluid nutrient medium, and inducible factor is 30-
60mg·L-1Sodium Pyruvate+10-50mgL-1Salicylic acid+0.1-0.3mgL-1Methyl jasmonate, vibrates under dark condition
Culture, once, subculture 3-5 times obtains liquid suspension culture cell to 3 weeks subcultures.Condition of culture is 25 ± 1 DEG C of temperature, shaking table
110-130 revs/min of rotating speed.
False-yellowflower milkwort root or herb suspension cell total saposins are separated and measure is comprised the following steps：False-yellowflower milkwort root or herb cell suspension cultures
Afterwards, it is centrifuged in centrifuge, filters out the cell in nutrient solution, be placed in constant temperature oven and dry to constant weight, is beaten with pulverizer
It is broken, it is put into round-bottomed flask, petroleum ether is added, filtered after the 3-4h that flowed back in 70-80 DEG C of water bath with thermostatic control, sunk obtained by filtering
Ethanol is added in shallow lake, and carries out ultrasonic wave extraction, extract solution is evaporated, after evaporation gained precipitation is dissolved with methyl alcohol, use first
Alcohol constant volume is to be measured.
Its total saponin content scope of false-yellowflower milkwort root or herb suspension cell is 0.72-0.9 mgg-1。
False-yellowflower milkwort root or herb cell culture method of the present invention prepares having the beneficial effect that for the method for saponins compound：
The present invention is using false-yellowflower milkwort root or herb callus of the plant tissue and cell culture production containing saponin compound higher and suspension
Cell, can not only be preserved the ecological environment with Economization on land, human resources, and not limited by extraneous factors such as seasons, can be with
Polygalic acid class compound medicine source is endlessly provided.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to
This.
Embodiment 1
1st, the acquisition of false-yellowflower milkwort root or herb aseptic seedling
The false-yellowflower milkwort root or herb seed collection time is September part, selects the sunny morning to gather whole infructescence, is cut fruit with scalpel
Under, uncracked fruit is chosen, with alcohol-pickled fruit 40-50 seconds of 70%, then with aseptic water elution 2 times, it is subsequently transferred to matter
Amount concentration of volume percent is to soak 10-12 minutes in 0.1% mercuric chloride solution, and with aseptic water washing 5 times, is inhaled with aseptic filter paper
Solid carbon dioxide point, is removed the pericarp of fruit with aseptic operation knife on aseptic operating platform, and seed is eliminated to be placed on aseptic filter paper
On, then the exosper of seed is removed, the seed of immature seed and maturation is separated, then immature seed accesses MS
+0.2mg·L-1 BAP（Benayl aminopurine）+ sucrose 30gL-1+ agar 5.5gL-1Culture medium in, ripe seed is accessed
In MS culture mediums, pH is 5.8-6.0.It is placed in 12 hd-1Photoperiod, 50 μm of olm of illumination-2 s-1, 25 ± 1 DEG C of temperature culturing room
Middle culture, mature seed starts to sprout for 10 days or so, and plumular axis and radicle are grown, and rear seedling is grown up within 30 days or so, immature seed 7
It or so forms adventitious bud, grows within 20 days or so adventitious bud clump, and breeding coefficient is about 10.
2nd, the culture of false-yellowflower milkwort root or herb callus and subculture
It is explant with organs such as the tender leaf of obtained aseptic seedling, plumular axis, stem apexs, described callus is aseptic false-yellowflower milkwort root or herb
Cultivated on inducing culture for explant and can obtain within 30 days callus, the optimal medium of callus induction in each position of seedling
It is MS+1.0 mgL-12,4-D（2,4 dichlorophenoxyacetic acid）+0.1mg·L-1TDZ（N- phenyl-N ' -1,2,3- thiadiazoles -
5- ureas）+ sucrose 30gL-1+ agar 5.5gL-1, pH is 5.8-6.0.In the callus same medium that will be obtained
After culture stabilization, average 3 weeks subcultures once, and squamous subculture 4-6 generations, can obtain pale yellow, loose callus.
3rd, false-yellowflower milkwort root or herb cell suspension cell culture
The callus that step 2 is obtained is inoculated in the triangular flask of the 100mL of 30mL fluid nutrient mediums, culture medium is：MS+
0.5 mg·L-12,4-D（2,4 dichlorophenoxyacetic acid）+0.1mg·L-1BAP（Benayl aminopurine）+ sucrose 30gL-1, pH is
5.8-6.0.Inducible factor is added in fluid nutrient medium can improve the content of total saposins in suspension cell, optimal inducible factor
It is 30-60mgL-1Sodium Pyruvate+10-50mgL-1Salicylic acid+0.1-0.3 mgL-1Methyl jasmonate, dark condition
Lower shaken cultivation, once, subculture 3-5 times obtains liquid suspension culture cell to 3 weeks subcultures.Condition of culture is 25 ± 1 DEG C of temperature,
110-130 revs/min of the rotating speed of shaking table.
4th, false-yellowflower milkwort root or herb callus and suspension cell total saposins are separated and determined
After false-yellowflower milkwort root or herb cell suspension cultures 20 days, 10min is centrifuged in the centrifuge of 2000rpm, in filtering out nutrient solution
Cell, be placed in 50 DEG C of constant temperature oven and dry to after constant weight, smashed with pulverizer, the callus of culture 30 days is equally put
Dried to constant weight in 50 DEG C of constant temperature oven, smashed with pulverizer, the callus after 0.5g is crushed and suspension are taken respectively
Cell sample is put into round-bottomed flask, adds 20mL petroleum ethers, is filtered after the 3-4h that flowed back in 70-80 DEG C of water bath with thermostatic control.
75% ethanol is added in filtering gained precipitation, and carries out ultrasonic wave extraction, ultrasonic frequency is high frequency（61KHz）, ultrasonic wave work(
Rate is 180W, each sample extraction twice, each 20-40min.Extract solution is evaporated, evaporation gained precipitation is molten with methyl alcohol
Xie Hou, it is to be measured in the volumetric flask of 25mL with methanol constant volume.With presenegenin as reference substance, 0.5mg is configured to methyl alcohol
mL-1Solution as reference substance solution, 575nm as wavelength is determined, through ultraviolet-visible spectrophotometer method measurement result, this
Its total saponin content scope of the preparation-obtained false-yellowflower milkwort root or herb suspension cell of inventive method is 0.60-0.85mgL-1, chrysanthemum
Omei meadowrue herb callus total saponin content 0.45-0.62mgg-1。
Embodiment 2
1st, the acquisition of false-yellowflower milkwort root or herb aseptic seedling
The false-yellowflower milkwort root or herb seed collection time is September part, selects the sunny morning to gather whole infructescence, is cut fruit with scalpel
Under, uncracked fruit is chosen, with alcohol-pickled fruit 40-50 seconds of 70%, then with aseptic water elution 2 times, it is subsequently transferred to matter
Amount concentration of volume percent is to soak 10-12 minutes in 0.1% mercuric chloride solution, and with aseptic water washing 5 times, is inhaled with aseptic filter paper
Solid carbon dioxide point, is removed the pericarp of fruit with aseptic operation knife on aseptic operating platform, and seed is eliminated to be placed on aseptic filter paper
On, then the exosper of seed is removed, the seed of immature seed and maturation is separated, then immature seed accesses MS
+0.5mg·L-1BAP（Benayl aminopurine）+ sucrose 30gL-1+ agar 5.5gL-1Culture medium in, ripe seed is accessed
In MS culture mediums, pH is 5.8-6.0.It is placed in 12 hd-1Photoperiod, 50 μm of olm of illumination-2 s-1, 25 ± 1 DEG C of temperature culturing room
Middle culture, mature seed starts to sprout for 10 days or so, and plumular axis and radicle are grown, and rear seedling is grown up within 30 days or so, immature seed 7
It or so forms adventitious bud, grows within 20 days or so adventitious bud clump, and breeding coefficient is about 8.
2nd, the culture of false-yellowflower milkwort root or herb callus and subculture
It is explant with organs such as the tender leaf of obtained aseptic seedling, plumular axis, stem apexs, described callus is aseptic false-yellowflower milkwort root or herb
Cultivated on inducing culture for explant and can obtain within 30 days callus, the optimal medium of callus induction in each position of seedling
It is MS+1.0mgL-12,4-D（2,4 dichlorophenoxyacetic acid）+0.1mg·L-1TDZ（N- phenyl-N ' -1,2,3- thiadiazoles -
5- ureas）+ sucrose 30gL-1+ agar 5.5gL-1, pH is 5.8-6.0.In the callus same medium that will be obtained
After culture stabilization, average 3 weeks subcultures once, and squamous subculture 4-6 generations, can obtain pale yellow, loose callus.
3rd, false-yellowflower milkwort root or herb cell suspension cell culture
The callus that step 2 is obtained is inoculated in the triangular flask of the 100mL of 30mL fluid nutrient mediums, culture medium is：MS+
0.5mg·L-12,4-D（2,4 dichlorophenoxyacetic acid）+0.1mg·L-1BAP（Benayl aminopurine）+ sucrose 30gL-1+ agar
5.5g·L-1, pH is 5.8-6.0.Inducible factor is added in fluid nutrient medium can improve the content of total saposins in suspension cell,
Optimal inducible factor is 30mgL-1Sodium Pyruvate+50mgL-1The mgL of salicylic acid+0.1-1Methyl jasmonate, it is dark
Under the conditions of shaken cultivation, once, subculture 3-5 times obtains liquid suspension culture cell to 3 weeks subcultures.Condition of culture is temperature 25 ± 1
DEG C, 110-130 revs/min of the rotating speed of shaking table.
4th, false-yellowflower milkwort root or herb suspension cell total saposins are separated and determined
After false-yellowflower milkwort root or herb cell suspension cultures 20 days, 10min is centrifuged in the centrifuge of 2000rpm, in filtering out nutrient solution
Cell, be placed in 50 DEG C of constant temperature oven and dry to after constant weight, smashed with pulverizer, be put into round-bottomed flask, 0.5g is added
20mL petroleum ethers, filter after the 3-4h that flowed back in 70-80 DEG C of water bath with thermostatic control.75% ethanol is added in being precipitated obtained by filtering,
And ultrasonic wave extraction is carried out, ultrasonic frequency is high frequency（61KHz）, ultrasonic power is 180W, each sample extraction twice, often
Secondary 20-40min.Extract solution is evaporated, after evaporation gained precipitation is dissolved with methyl alcohol, with methanol constant volume in the volumetric flask of 25mL
In it is to be measured.With presenegenin as reference substance, 0.5mgmL is configured to methyl alcohol-1Solution as reference substance solution, 575nm
As wavelength is determined, through ultraviolet-visible spectrophotometer method measurement result, the preparation-obtained false-yellowflower milkwort root or herb of the inventive method
Its total saponin content scope of suspension cell is 0.7-0.9mgg-1。
With presenegenin as reference substance, 0.5mgmL is configured to methyl alcohol-1Solution as reference substance solution, 575nm
As wavelength is determined, through ultraviolet-visible spectrophotometer method measurement result, the preparation-obtained false-yellowflower milkwort root or herb of the inventive method
Its total saponin content scope of suspension cell is 0.72-0.9mgg-1。
MS in the present invention（Murashige and Skoog）It is international culture medium, its composition and compound method
Referring to following documents：Li Jun is bright, and Zhu steps on cloud plant cell culture technologies（3rd edition）[M] Beijing：China Agricultural University publishes
Society, 2006., using high-temperature sterilization pot sterilizing, sterilize 20min at 0.105MP and 121 DEG C for culture medium and sterilized equipment used.
The method of the present invention can produce the secondary metabolite of false-yellowflower milkwort root or herb, low production cost, not by season and
Environment is limited, cycle is short.
Above-described embodiment is only used for illustrating inventive concept of the invention, rather than the restriction to rights protection of the present invention, all profits
The change that unsubstantiality is carried out to the present invention is conceived with this, protection scope of the present invention all should be fallen into.
Claims (6)
1. a kind of method that false-yellowflower milkwort root or herb cell culture method prepares saponins compound, it is characterised in that comprise the following steps：
（1）The acquisition of false-yellowflower milkwort root or herb seed；
（2）The sterilization of false-yellowflower milkwort root or herb seed and the acquisition of aseptic seedling；
（3）Induction false-yellowflower milkwort root or herb Callus formation；
（4）False-yellowflower milkwort root or herb callus is carried out into squamous subculture, the callus of loose stabilization is obtained；
（5）Callus is transferred in fluid nutrient medium carries out shaken cultivation；
（6）The inducible factor of addition variety classes and various concentrations, filters out high yield cell；
（7）High yield suspension cell is carried out into Multiplying culture；
（8）The drying and crushing of callus or suspension cell；
（9）The extraction of total saposins, purifying and measure in callus or suspension cell.
2. the method that false-yellowflower milkwort root or herb cell culture method as claimed in claim 1 prepares saponins compound, it is characterised in that institute
State step（2）Middle false-yellowflower milkwort root or herb aseptic seedling is comprised the following steps：Fruit is cut with scalpel, is chosen uncracked
Fruit, with alcohol-pickled fruit 40-50 seconds, then uses aseptic water elution, and it is 0.1% to be subsequently transferred to quality concentration of volume percent
Soaked 10-12 minutes in mercuric chloride solution, and with aseptic water washing, use aseptic filter paper suck dry moisture, nothing is used on aseptic operating platform
Bacterium scalpel removes the pericarp of fruit, and seed is eliminated to be placed on aseptic filter paper, then the exosper of seed is removed, will
The seed of immature seed and maturation is separated, and then immature seed accesses MS+0.2-0.5mgL-1Benayl aminopurine+
Sucrose 30gL-1+ agar 5.5gL-1Culture medium in, ripe seed is accessed in MS culture mediums, and pH is 5.8-6.0, is placed in
12hd-1Photoperiod, 50 μm of olm of illumination-2 s-1, 25 ± 1 DEG C of temperature culturing room in cultivate, mature seed starts to sprout for 10 days or so
Hair, plumular axis and radicle are grown, and rear seedling is grown up within 30 days or so, and immature seed forms adventitious bud in 7 days or so, grows within 20 days or so
Adventitious bud clump, breeding coefficient is about 8-10.
3. the method that false-yellowflower milkwort root or herb cell culture method as claimed in claim 1 prepares saponins compound, it is characterised in that yellow
The induction of flower omei meadowrue herb callus and subculture are comprised the following steps：It is with organs such as the tender leaf of obtained aseptic seedling, plumular axis, stem apexs
Explant or with adventitious bud as explant, described callus is each position of false-yellowflower milkwort root or herb aseptic seedling for explant or with not
Normal bud is cultivated on inducing culture for explant and can obtain within 30 days callus, and the culture medium of callus induction is MS+1.0
mg·L-12,4 dichlorophenoxyacetic acid+0.1mgL-1N- phenyl-N ' -1,2,3- thiadiazoles -5- ureas+sucrose 30gL-1+
Agar 5.5gL-1, pH is 5.8-6.0, after the callus that will be obtained is stablized with same medium culture, average 3 weeks subcultures
Once, and squamous subculture 4-6 generations, pale yellow, loose callus can be obtained.
4. the method that false-yellowflower milkwort root or herb cell culture method as claimed in claim 1 prepares saponins compound, it is characterised in that yellow
Flower omei meadowrue herb cell suspension cell culture is comprised the following steps：The callus of acquisition is inoculated in the triangular flask of fluid nutrient medium
In, culture medium is：MS+0.5 mg·L-1 2,4 dichlorophenoxyacetic acid+0.1mgL-1Benayl aminopurine+sucrose 30gL-1+
Agar 5.5gL-1, pH is 5.8-6.0, and inducible factor is added in fluid nutrient medium, and inducible factor is 30-60mgL-1Acetone
Sour sodium+10-50mgL-1Salicylic acid+0.1-0.3mgL-1Methyl jasmonate, shaken cultivation under dark condition, 3 weeks subcultures
Once, subculture 3-5 times, obtains liquid suspension culture cell；Condition of culture is 25 ± 1 DEG C of temperature, the rotating speed 110-130 of shaking table
Rev/min.
5. the method that false-yellowflower milkwort root or herb cell culture method as claimed in claim 1 prepares saponins compound, it is characterised in that yellow
Flower omei meadowrue herb callus or suspension cell total saposins are separated and measure is comprised the following steps：False-yellowflower milkwort root or herb cell suspension cultures
Afterwards, it is centrifuged in centrifuge, filters out the cell in nutrient solution, the callus or suspension cell that will be obtained is respectively placed in perseverance
Dried to constant weight in warm baking oven, smashed with pulverizer, be put into round-bottomed flask, petroleum ether is added, in 70-80 DEG C of thermostatted water
Filtered after backflow 3-4h in bath, ethanol is added in being precipitated obtained by filtering, and carry out ultrasonic wave extraction, extract solution is steamed
Hair, it is to be measured with methanol constant volume after evaporation gained precipitation is dissolved with methyl alcohol.
6. the method that false-yellowflower milkwort root or herb cell culture method as claimed in claim 1 prepares saponins compound, it is characterised in that：It is yellow
Flower omei meadowrue herb suspension cell or its total saponin content scope of callus are 0.72-0.9 mgg-1。
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| CN109302987A (en) * | 2018-11-16 | 2019-02-05 | 闽南师范大学 | A kind of abductive approach of false-yellowflower milkwort root or herb callus |
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